Introduction

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening hereditary disorder, affecting approximately 600,000 Americans and over 12 million people worldwide (1–3). Mutations in polycystic kidney disease 1 (PKD1) and PKD2, which encode polycystin-1 and polycystin-2, account for approximately 85% and approximately 15% of the disease, respectively. The hallmark of ADPKD is the development and relentless growth of innumerable kidney cysts that ultimately result in loss of kidney function by age 60 years old in 50% of patients (1,3,4).

FGF23 is a powerful risk factor for cardiovascular events and all-cause mortality across the spectrum of CKD (10–14). Few studies (15,16) in adults and one in children (17) have reported an independent association between higher FGF23 concentrations and incidence and progression of kidney disease in patients with an eGFR≥60 ml/min per 1.73 m2. Another study found no association between FGF23 and risk of incident CKD in patients with type 2 diabetes (18). Similarly, studies of FGF23 and kidney disease progression in patients with eGFR<60 ml/min per 1.73 m2 have also yielded contradictory results (11,12). These studies included community-based general nephrology patients with high rates of cardiovascular disease or other comorbidities, which are important confounders of the potential association of FGF23 with kidney function decline, and none included patients with ADPKD. Because individuals with ADPKD tend to have fewer comorbid conditions than those with CKD due to other etiologies, adult patients with ADPKD are a unique population in whom to investigate the effects of FGF23 on CKD progression. In addition, little is known about alterations in mineral metabolism in ADPKD (19).

To date, no studies have examined the relationships between FGF23 and kidney disease progression in individuals with ADPKD across the spectrum of eGFR (19). Because increases in height-adjusted total kidney volume (HtTKV) reliably predict the rate of decline in kidney function and risk of kidney failure in ADPKD (20), HtTKV is an especially powerful biomarker of disease progression in patients with early stages of ADPKD and normal or nearly normal kidney function (21). We tested the hypothesis that, in participants of the HALT-PKD (22–24) Study, elevated FGF23 concentrations are associated with progression of ADPKD marked by reductions in eGFR, increases in HtTKV, and incident ESRD. In addition, we determined if circulating FGF23 levels measured at baseline can improve risk prediction in patients with ADPKD enrolled in the HALT-PKD Study.

Study A (23) enrolled 558 patients who were hypertensive with ADPKD ages 15–49 years old with an eGFR>60 ml/min per 1.73 m2 (2), and Study B enrolled 486 patients with ADPKD ages 18–64 years old with eGFR of 25–60 ml/min per 1.73 m2 (24). In Study A, the primary outcome was the annual percentage change in HtTKV measured by magnetic resonance imaging (MRI). In Study B, the primary outcome was the composite end point of time to 50% reduction of baseline eGFR, ESRD (initiation of dialysis or preemptive transplant), or death. All participants from the HALT-PKD Study A and Study B with available serum samples were included in these analyses, resulting in a final cohort of 1002 participants (Study A =540; Study B =462). The institutional review board at the University of Colorado Denver and the HALT-PKD Study Executive Steering Committee approved this ancillary study.

Exposure Variable

All participants provided stored serum samples at their baseline visit, which were stored in a central repository at −80°C until they were shipped to the University of Washington for analyses.

The primary exposure variable for this analysis was serum intact FGF23. Intact FGF23 was measured in duplicate using the Kainos immunoassay, which detects the full-length, biologically intact FGF23 molecule via midmolecule and distal epitopes. The intra- and interassay coefficients of variability are 3.8% and 3.0%, respectively, for this assay.

Outcomes

Reflecting termination of follow-up due to death or transplant, the follow-up duration for mortality was a median of 5.4 years. Classifications of outcomes were made at the clinical centers and then reviewed by an outcome committee composed of the HALT-PKD Study investigators who were blinded to the randomized treatment assignments (22). The outcomes for this analysis were decided a priori: (1) annualized change in eGFR in Study A and Study B, (2) mean annualized percentage change in HtTKV in the Study A participants who underwent an MRI, and (3) time to 50% reduction of baseline eGFR, ESRD (initiation of dialysis or preemptive transplant), or death or their composite in Study B participants. MRI images were obtained at each participating clinical center at baseline and 24, 48, and 60 months using a protocol developed by the HALT-PKD Study Imaging Subcommittee. After acquisition, MRI images were transferred securely to the Image Analyses Center at the University of Pittsburgh (22).

Covariates

Age, sex, race, body mass index (BMI), PKD genotype, and comorbid medical conditions were recorded during the baseline phase of the trial. PKD genotype was categorized as PKD genotype 0 (unknown), PKD genotype 1 (PKD1), and PKD genotype 2 (PKD2). The eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) prediction equation (25) on the basis of serum creatinine measured by a centralized HALT-PKD Study laboratory at baseline, at the 4- and 12-month visits, and every 6 months subsequently. BP measurements were standardized across participating clinical centers. All participants had their BP measured three times while seated and once while standing (22). A 24-hour urine collection was performed at baseline and annually in the HALT-PKD Study A and Study B. Urine albumin excretions were determined at the Diagnostic Laboratory Facility at Brigham and Women’s Hospital (Boston, MA) (22). Serum calcium and phosphorus were measured annually at the Cleveland Clinic central laboratory using standard techniques.

Statistical Analyses

Demographics, cardiovascular risk factors, and markers of mineral metabolism were compared across quartiles of baseline serum FGF23 levels using the chi-squared test for categorical factors and ANOVA for continuous variables, with log transformations applied to variables with heavily right-skewed distributions.

We used linear mixed effects models to examine the association between baseline FGF23 levels and mean annualized change in eGFR and mean annualized percentage change in HtTKV as was previously done (23,24). FGF23 was right skewed and therefore, modeled in quartiles. All linear mixed effects models included a random intercept for each participant and a random slope for time as a continuous variable to account for within-participant correlation. For all the linear mixed effects models, we determined the P values for trend across the FGF23 quartiles.

We used Cox proportional hazards models to examine the risks of 50% reduction in eGFR from baseline, ESRD, death, or their composite according to baseline FGF23 levels expressed as a continuous variable with hazard ratios calculated per doubling of FGF23 and in quartiles, with the lowest quartile defined as the reference group. We used a combination of criteria on the basis of the HALT-PKD Study design, subject matter considerations, and biologic understanding to designate a nested set of covariates for inclusion in all models. In the final multivariable models, we adjusted for age, sex, randomization group, and baseline measurements of systolic BP, BMI, eGFR, urinary albumin excretion, serum calcium, phosphorus, and PKD genotype.

We examined the incremental value of adding FGF23 to logistic regression models, examining a relationship between HtTKV as a continuous variable and an annualized change in eGFR≥3 ml/min per 1.73 m2 in Study A participants by calculating a C statistic (26). A change in eGFR≥3 ml/min per 1.73 m2 per year is an established cutoff to define rapid progression in ADPKD (20,21,23,27). We limited these analyses to Study A participants, because HtTKV data were only available in Study A.

We confirmed no violation of the proportional hazards assumption using Schoenfeld residuals on functions of time. Two-tailed values of P<0.05 were considered statistically significant without formal adjustment for multiple comparisons. All statistical analyses were performed with SAS software, version 9.4 (SAS Institute, Cary, NC).

Results

We measured serum intact FGF23 in 1002 participants with ADPKD from the HALT-PKD Study, including 540 from Study A and 462 from Study B. At baseline, participants in Study A had a mean age of 37±8 years old, mean eGFR of 91±17 ml/min per 1.73 m2, median HtTKV by MRI of 589 (interquartile range, 405−860) ml/m, and median FGF23 of 44 (33−56) pg/ml. In Study A participants, higher FGF23 concentrations were significantly associated with men, lower eGFR, and higher systolic BP, BMI, and HtTKV (Table 1). Participants in Study B had a mean age of 49±8 years old, mean eGFR of 48±12 ml/min per 1.73 m2, and median FGF23 level of 69 (interquartile range, 50–93) pg/ml (the HALT-PKD Study B did not include an MRI). Higher FGF23 concentrations were mainly associated with a lower eGFR and higher serum phosphorus and urine albumin excretion in Study B participants (Table 2).

Associations of baseline fibroblast growth factor 23 concentrations with annualized percentage change in height-adjusted total kidney volume in the HALT-PKD Study A

FGF23 and Hard End Points

During a median follow-up of 5.4 years, 36% (n=175) of participants experienced a ≥50% reduction of baseline eGFR, 22% (n=108) reached ESRD, and six participants died, of whom only three died before reaching ESRD. Ascending FGF23 quartiles were associated with higher risk of the composite outcome of ≥50% reduction of baseline eGFR, ESRD, or death in unadjusted analyses and analyses that adjusted for demographics, comorbidities, baseline kidney function, other variables of mineral metabolism, randomization group, and PKD genotype (Table 5). In the fully adjusted models, the risk of developing a kidney outcome or death was more than threefold higher in the fourth versus the first quartile. Similar results were observed when each of the end points was analyzed separately. Results remained unchanged when FGF23 was modeled continuously (Table 5). Finally, we detected no significant interactions between FGF23 and PKD genotypes for all hard kidney end points (P for all interactions >0.30).

Associations of baseline fibroblast growth factor 23 concentrations with risk of 50% decline from baseline eGFR, ESRD, or the composite end point in the HALT-PKD Study B

Risk Prediction

Lastly, we examined the addition of circulating FGF23 levels to logistic regression models, testing the association between HtTKV as a continuous variable and an annual eGFR decline ≥3.0 ml/min per 1.73 m2 as the outcome. As depicted in Table 6, addition of FGF23 to unadjusted and adjusted models did not improved the C statistic, suggesting that, in Study A participants, the capacity of HtTKV to predict rapid kidney function decline progression was not enhanced by addition of FGF23.

Association of height-adjusted total kidney volume with annual decline in eGFR≥3 ml/min per 1.73 m2 per year

Discussion

Our study shows that higher serum FGF23 was independently associated with risk of kidney disease progression in patients with early and later stages of ADPKD. We found significant associations between higher baseline serum FGF23 and longitudinal rates of eGFR reduction in all study participants, including those with an eGFR>60 ml/min per 1.73 m2. Higher baseline FGF23 was also independently associated with accelerated increases in longitudinal HtTKV, which is an established marker of ADPKD progression in patients with early-stage disease (20). Among participants with advanced disease at baseline, participants with FGF23 levels in the highest quartile were at threefold increased risk of developing clinically relevant events, including more rapid time to 50% reduction from baseline eGFR and onset of ESRD or death. Among participants with preserved eGFR at baseline, the highest quartiles of FGF23 were associated with clinically significant declines in eGFR and clinically meaningful increases in HtTKV (20,21,23,27). However, the addition of FGF23 to a model that adjusted for traditional risk factors, did not improve risk prediction of rapid kidney function decline.

Established risk factors for ADPKD progression include PKD1 mutations (28), men (29), early onset of hypertension (30,31), early onset of repeated episodes of gross hematuria (32), greater HtTKV (20), early decrease in kidney function (21,33), presence of microalbuminuria, and elevated serum copeptin levels (34–36). Until now, FGF23 has never been examined as a risk factor for disease progression in ADPKD (19). In fact, little is known about mineral metabolism dysregulation in ADPKD (19). In a cross-sectional study, FGF23 levels were higher in patients with ADPKD (mean eGFR =100 ml/min per 1.73 m2) than in eGFR-matched patients with CKD due to other causes (37). In the ADPKD group, serum phosphorus levels were significantly decreased, whereas circulating parathyroid hormone levels were normal, suggesting that elevated FGF23 was biologically active and exerting physiologically relevant phosphate wasting early in the course of ADPKD (37). These findings added to the observations by Spichtig et al. (38) that both FGF23 mRNA and protein are detected in the cells lining renal cysts in animal models of PKD have intrigued the ADPKD research community about the role of FGF23 in ADPKD-related clinical outcomes. Hence, our findings suggest that, although higher circulating FGF23 levels were associated with kidney function decline and HtTKV changes, FGF23 did not outperform current predictors of rapid kidney disease progression in patients with ADPKD and normal kidney function.

Although our predictive analyses do not support the use FGF23 as a biomarker of rapid kidney function decline in patients with early ADPKD, our findings of independent associations between elevated FGF23 levels and ADPKD-related outcomes in Study A and Study B raise important questions about a potential mechanistic role of elevated FGF23 in ADPKD progression. A plausible hypothesis that requires further study could be that high FGF23 levels, reflecting either dysregulated FGF23 synthesis by osteocytes triggered by a defect in the primary cilium/polycystin 1 and 2 complex or ectopic secretion of FGF23 by kidney tubular cells, exert profibrotic or toxic effects on kidney tubules or other structures (19,38).

There are several limitations in this study. First, this observational study cannot establish a causal relationship between higher serum FGF23 concentrations and kidney disease progression in ADPKD. Second, we cannot rule out the possibility of residual confounding. Third, the level of kidney function was defined by the estimated CKD-EPI prediction equation rather than more direct measures, such as iothalamate or inulin clearance data. Fourth, most of the participants in this study were white; therefore, caution should be used when extrapolating these results to other populations. Fifth, our findings are on the basis of a single baseline measurement of FGF23 to predict ADPKD outcomes; whether longitudinal changes in FGF23 concentrations add incremental predictive value is unknown.

Our study also has considerable strengths. Foremost, this is the first study to our knowledge to evaluate the association of serum FGF23 with HtTKV and kidney function decline across a wide spectrum of kidney function in ADPKD. Other strengths include the relatively large cohort, complete nature of the dataset, and the long durations of follow-up. In fact, the HALT-PKD Study (22–24) provided a unique opportunity to use high-quality data derived from randomized clinical trials to prospectively examine FGF23 across the spectrum of eGFR. Importantly, we were able to statistically adjust for multiple factors that affect kidney disease progression in ADPKD, such as microalbuminuria. Finally, all measurements of serum FGF23 were performed in a standardized fashion in a single reference laboratory.

In conclusion, we determined that higher serum FGF23 concentrations were associated with changes in HtTKV, kidney function decline, onset of ESRD, and death in patients with ADPKD across the spectrum of kidney function. However, FGF23 did not improved prediction of rapid kidney function decline in patients with early disease ADPKD. Further studies are necessary to determine the extent to which our results represent causal effects of FGF23 on progression (for example, by promoting tubulointerstitial fibrosis). If FGF23 plays a causal role in PKD progression, other studies should test whether interventions that reduce FGF23 concentrations might prove to be renoprotective in ADPKD.

Disclosures

None.

Acknowledgments

This research was supported by grant DK090005-01A1 from the National Institute of Diabetes and Kidney Diseases.

Because M.C. is a Deputy Editor of the Clinical Journal of the American Society of Nephrology, he was not involved in the peer review process for this manuscript. Another editor oversaw the peer review and decision-making process for this manuscript.

Footnotes

Published online ahead of print. Publication date available at www.cjasn.org.